The goal of this project is to develop a selection strategy using RNA interference (RNAi) to identify novel therapeutic targets for the treatment of cytolytic virus infection. RNAi is a cellular process that causes targeted elimination (silencing) of mRNA. Nucleonics, Inc. has developed platform technologies to exploit RNAi for development of therapeutics and genomic applications. These technologies provide powerful new tools for targeted elimination of specific mRNAs. RNAi will be used to selectively silence genes required for cytolytic virus replication thereby producing cells that will be resistant to infection. Repeated rounds of selection (infection with cytolytic virus) and enrichment (isolation of RNAi-inducing sequences in surviving cells) will identify genes that are potential therapeutic targets for treatment of virus infection. Phase I of this application focuses on ( i ) optimizing vectors and delivery systems for inducing RNAi in mammalian cells and (ii) "proof-of-concept" tests using these vectors and delivery systems to silence viral and cellular genes known to be required for cytolytic virus replication. Two cytolytic viruses will be tested in this system, a DNA containing virus, human herpes simplex virus type I (HSV-1), and an RNA containing virus, bovine viral diarrhea virus (BVDV). BVDV is a tissue culture surrogate for hepatitis C virus. Phase II of this proposal focuses on using this selection to identify novel genes required for cytolytic virus infection and validate these novel targets in cell culture and animal models of infection.